Historical Overview of the First Two Waves of ... - ACS Publications

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Downloaded by UNIV OF ARIZONA on December 9, 2012 | http://pubs.acs.org Publication Date (Web): December 7, 2012 | doi: 10.1021/bk-2012-1119.ch006

Historical Overview of the First Two Waves of Bactericidal Agents and Development of the Third Wave of Potent Disinfectants J. Liu,1,2 K. Chamakura,3 R. Perez-Ballestero,3 and S. Bashir*,4 1Department

of Chemistry, Texas A&M University-Kingsville, MSC 161, 700 University Blvd., Kingsville, Texas 78363, U.S.A. 2Department of Chemistry, Texas A&M University, P.O. Box 30012, College Station, Texas 77842-3012, U.S.A. 3Department of Biological and Health Sciences, Texas A&M University-Kingsville, MSC 158, 700 University Blvd., Kingsville, Texas 78363, U.S.A. 4Chemical Biology Research Group, Texas A&M University-Kingsville, MSC 161, 700 University Blvd., Kingsville, Texas 78363, U.S.A. *E-mail: [email protected]

Certain microorganisms could cause disease and morbidity. Disinfection science was used as a “shield” for protection of human health, including access and availability to clean water. Two broad strategies were used, physical separation, purification, chemical inhibition, and sterilization. In the field of chemical disinfection, three chemicals were widely used, alcohols, phenols and bleach. Adjunct to use of chemical disinfectants was the use of metals or metal oxides, such as silver or titania. Silver has been used for years and has recently been re-evaluated due to the advent of nanotechnology. Silver nanoparticles have been extensively used in the treatment of disease and purification and heralded the ‘second wave’ of disinfection science, the ‘first wave’ being physical and chemical disinfection based on organic and inorganic compounds or salts. Recent advances in structured or engineered surfaces like zeolites has enabled ultrahigh surface area and rapid sterilization through the use of metal organic

© 2012 American Chemical Society In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

frameworks (MOFs) as the ‘third wave’ of disinfection science. MOFs offer the same advantages as colloids but also have ultra-high surface area, long-term persistence and ultra-low doses, which will be applied toward water purification.


Prelog: Disinfection Science In this book chapter, only chemical disinfectants of bacteria will be discussed in the form of supplemental tables and summaries; with a brief overview of physical methods followed by chemical in general followed by halogen/ozone/hydrogen peroxide and finally use of metal nanoparticles and comparison with our current results. Following the introduction, a brief experimental design to assess bleach and phenol and silver nanoparticles will be described including characterization methods. The related results, advantages and disadvantages of each type of disinfectant will be stated, followed by a comparison with current literature. The context of the results will be discussed in terms of mechanisms of inhibition and our findings will be compared and contrasted with other literature findings of similar systems. Lastly, a new approach using metal organic frameworks will be briefly described and concluded with future trends in the area of disinfection science with particular emphasis of use of metal/metal-oxide nanoparticles and metal organic framework based disinfectants towards water purification, surface sterilization and possible uses in health care.

Introduction Introduction to Disinfection Science: A Historical Overview Between 1840-1857, three scientists, Ignaz Semmelweis (1), Louis Pasteur (2) and Joseph Lister (3) had demonstrated that microbes could cause disease and that disinfection through chemical means such as with bleach (Semmelweis used a mixture of calcium chloride hypochlorite, calcium hypochloride and calcium chloride) or heat treatment (pasteurization), or bandages treated with phenol greatly reduced the transmission of disease. Robert Koch (between 1876-1884) has set the foundation of the science of microbiology and evaluation of chemicals that could be used toward disinfection or disease-control as demonstrated by Rideal and Walker (4). The approach required for “standard “ in vitro “procedures (5)” using specific strains such as Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 15422) using the proscribed dilution method (6), although other microorganism were also evaluated such as Escherichia coli (7). These protocols firmly established the profound benefits of sterilization (for inanimate matter (8)) and antisepsis (for living matter) (9). As early as 1900, a review of the literature in a report to the committee on disinfectants identified the following candidates: formaldehyde, mercuric chloride, chloride of lime [calcium (hydroxide/chloride/hypochlorite)], sulphurous acid, phenol, copper sulphate, zinc chloride, quick lime [calcium oxide ] and boiling of water (10), some of which are used today, which relay on chemical or physical methods (11–15) towards disinfection. 130 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


The effectiveness of a particular treatment has been quantified by using the decimal reduction time (D value, in units of time) defined as the time at fixed temperature and pressure required for one log reduction of a the initial colony forming units, for example plotting colony forming units (CFUs) versus time, the rate of decrease can be calculated. If the log decrease in CFU is repotted against time, the D value can be directly obtained from the gradient as the negative reciprocal. If the D value for the same microbe is plotted against changes in temperatures at fixed pressure, the “sensitivity” of the microbe to heat can also be determined, this value has been dubbed the Z value (in units of temperature) (16). The time and temperature for disinfection is dependent on the type of microbe, its growth cycle and with microbial spores requiring higher temperatures (17). The actual temperature/pressure profile is related to type of media (for example whether milk, fruit juice, alcohol is being used), but one general method that could apply to a broad range of food stuffs, medical equipment or storage containers is rapid heating at 150 °C for 10 seconds under steam convection or heating at 75 °C and cooling for an extended period of time for temperature-sensitive liquids, fabrics or equipment (18) by following standard operation procedures, such as outlined by the food and drug administration or other regulatory agencies (19).

The First Wave Disinfectant: A Historical Overview Chemical disinfectants are chemicals that inhibit and inactivate microbes. Since microbes are living biological organism, which may use oxygen as their terminal electron acceptor (aerobic respiration) or other, small molecules in the absence of oxygen (anaerobic respiration), possible intervention at physical or electrochemical disruption can lead to inhibition of growth and cellular inactivation. Inhibition can be achieved chemically, the specific disinfectant classified based on the functional group or mode of inhibition e.g. acid, base, detergent, respiration inhibitor respectfully, following published guidelines, set forth by the federal government or professional associations, such as association for professionals in infection control and epidemiology (20). Citric fruit, such as oranges are known to spoil slowly unlike other fruits such as bananas and reason for this is thought to be due to citric acid, which has been used as a disinfectant. The principal advantages of acid based disinfectants are speed of action (almost immediate) and lower costs and negligible toxicity to humans (for weak acids). The halogens (21–25) are potent oxidants, with chlorine being the most commonly used oxidants from the family. Chlorine, unlike iodine is a gas at room temperature and as a disinfectant is in the form of hypochlorite or hypochlorous anion, the most widely used being as the sodium salt of hypochlorite. As in the case of iodine, the disinfectant ‘strength’ is related to concentration of the oxidizing agent, whether the agent is in ‘free’ form, or whether it is slowly release as was shown for poviodine-iodine or as chloramines for release of chlorine. Sodium hypochlorite (bleach) is light yellow liquid with 5 % chlorine. Bleach is probably one of the most widely used forms of disinfection of water, medical equipment and households in the ppm range. 131 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Peroxides (26) are compounds with the oxidation state for oxygen of minus one, are diamagnetic, and are similar to superoxide and ozonides, which have the same oxidation, state, and unlike peroxides are paramagnetic and prone to radical ion formation. Examples of peroxides include hydrogen or benzoyl peroxide, which is a strong oxidizing agent and is used in disinfection and sterilization including wound infection. Hydrogen peroxide is generally obtained as a dilute solution (< 5%) and rapidly decomposes and comes in a formulation with other chemical additives as stabilizers towards decomposition. Ozone is a gas which is known to absorb UV radiation, but can be used in the formation of reactive oxygen species including hydroxide radical and hydroperoxyl (HO2) radicals, including peroxide and superoxide (oxidation state minus one-half), which cause microbial lysis. The peroxides are generally used at concentrations of less than 5% and slightly higher percentage as a bleaching agent, with higher percentages used for sterilization. As a disinfectant, hydrogen peroxide like bleach is fast acting and used in the ppm range, to reduce the microbial population by 90% (decimal reduction time, D) within minutes and may be used in gaseous or vapor form for disinfection of entire rooms. For disinfection of water, ozone is used in less than 1 ppm, with hydrogen peroxide in the low ppm is effective more against Gram-positive than Gram-negative microbes. The problem with these disinfectants are disinfection by-products, possible corrosion of the container, such as iron containers, slow acting of benozyl peroxide due to its low solubility in water or bleaching effect of hydrogen peroxide. Phenol, one of the oldest chemical disinfectants (27, 28) was used as an antiseptic spray was by Joseph Lister (1900s) in the form of triclosan, a mixture of salicylic acid and chloroxylenol and is a stronger acid than aliphatic alcohols. The formulation used is in the form of a micelle or detergent and are applied to a wide array of microbes and surfaces, generally in low ppm. Phenol itself is water-soluble and used as a standard disinfectant to which new forms of disinfectants are compared to in terms of D-value of phenol (P value coefficient). The general effectiveness of phenol as a disinfectant is against Gram-positive microbes. Since phenol or phenol-based disinfectants have been used in many settings for over a century, with over usage has led to some microbial resistance. Salicylic acid is a co-additive to the above, which is also an anti-inflammatory agent, in the form of antimicrobial soaps or detergents or cleaning agents, usually at 1000 ppm, have like phenols been used against a wide variety of microbes, particularly Gram-positive microbes (21–23, 26–29).

Mechanism of Inhibition of Chemical Disinfectants The above introduction to chemical disinfectants (30–32) can be considered the ‘first wave’ and have been shown to be effective for a wide-variety of uses including disinfection and sterilization. Review of Figure 1 indicates that inhibition is achieved through a number of different processes discussed above. Briefly, physical destruction of the cell wall, membrane or macromolecules can be achieved, or oxidation of cellular machinery such as nucleic acids or proteins or membrane glycoproteins would cause disruption of critical systems. Other 132 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


processes are mechanical disruption of certain regions, such as introduction of cellular pores, cross-linking of amino acids or nucleotides would cause disruption and microbial inactivation. The mechanism (33–43) by which acids inhibit microbe are varied, but can be generalized as follows: Strong acids (33) lead to total dissociation and a lower pH, the low acidic environment disrupts the electrical membrane potential (due to disruption of proton motive force), hijacking the normal machinery of the microbe for nutrient uptake and storage, in addition to modification and potential oxidation of surface lipids or proteins. For weak acids, the lowering of the pH is not an issue, since the pH is between 4-5, which may also be sufficient to disrupt the membrane potential, uptake, storage and utilization of essential nutrients, leading to disruption of ATP. Other disruptions may include mis-match in macromolecule synthesis, such as where proteins or nucleic acid are covalently cross-linked at specific amino acids, such as cysteine. This linking will eventually leading to disruption of oxidation phosphorylation and inhibition of respiration particularly with ester derivatives of the acids that may interaction with the microbial lipid structures more efficiently than acids. With bases, partial solubilization (37) of the membrane may occur due to occurrence of phospholipids, lipids and fatty acids. Disruption of the cell wall (for Gram-negative microbes) and plasma membrane will cause lysis, protein degradation, similar to processes involved with acids. Alcohols (34) assist with protein denaturation, cross-linking through hydrogen bonding and membrane lysis (35). Anilides and Diamides class (36) of disinfectants such as tribromsalan and propamidine respectfully are effective against Gram-positive bacteria (37), but have limited use as disinfectants. The former class inhibit through interaction with the plasma membrane leading to disruption of the lipid bilayer. The latter have been shown to be effective against gram-positive bacteria. The mode of action of peroxides is through oxidation of the membrane or macromolecules, for example for chlorine the active species is the hypochlorous anion. The targets for oxidation are sulfhydryl groups in cysteine amino acids, and oxidation of other amino acids in general, membrane lipids, sugars and membrane proteins respectfully affecting oxidative phosphorylation leading to cell death. Oxidation can be achieved directly through donation (transfer) of oxygen, or acceptance of electrons. Since this process is mutually exclusive, the electron acceptor becomes reduced and the electron donor becomes oxidized. Common examples include the halogens (38), producing halogen anion (or trianion for iodine); mineral acids producing the oxide/dioxide; bleach, producing water and the halide anion; oxygen producing oxides, water and carbon dioxide; ozone producing oxidation by-products and water and permanganate producing the metal cation and/or metal oxide’ and peroxides producing oxides and water (39–42). Phenols (43) are known to increase the cell wall permeability through interaction with cell wall enzymes via the hydroxyl moiety of the parent molecule, leading to ion leakage and membrane lysis. Increased permeability will disrupt the membrane potential due to uncoupling of proton motive force and oxidative phosphorylation, in addition to disruption of key kinases, isomerases and decarboxylases. One example is that triclosan is known to inhibit enoyl reductase, 133 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


in addition to membrane insertion into lipid components (58). Triclosan is a heterodimer of dichlorophenoxy and chlorophenol, while hexachlorophene and chloroxylenol are other derivatives, which have been used. Lastly, since all living cells required chemical energy to reproduce, grow or undergo regeneration or repairs, chemical energy is obtained indirectly from raw materials via redox processes (44) or directly from sunlight, also using redox processes (45), albeit of a different kind and any disruption of these processes would be vital (46). These modes of action are in five groupings and are summarized in Figure 1.

Figure 1. Chemical disinfectants interact through many avenues. Five general processes are: (1) pH-assisted disruption of membrane phospholipids/peroxidation leading to membrane rupture; (2) disruption of proton motive force leading to loss of membrane permeability and leakage of vital ions; (3) membrane-insertion, denaturation and loss of function; (4) oxidation or covalent of critical enzymes (star) such as enoyl reductase, kinases, decarboxylases and/or covalent modification of topoisomerases, DNA Intercalation (○, e.g at the minor groove) leading to loss of function, cellular shutdown; and (5) disruption of ATPase, oxidation phosphorylation leading to inhibition of respiration.

134 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


The Second Wave: A Historical Overview Recently a newer approach as the ‘second wave’ has been evaluated in the last four decades, using metal or metal oxides as disinfectants (47–50). Metals can be group into three categories, metals that in trace amounts are essential for metabolic processes, metals with in higher amounts are toxic and metals, which are toxic and not involved in biochemical, processes (51). Copper has historically been used by bakers as a sterilization agent in chimney fumes to inactivated any yeast and when accumulated can be toxic. Silver has been used in ancient times either as metal silver, or in the form of 1% silver sulfadiazine solution to prevent wound infection (52) or surface sterilization. Compared to their chemical disinfectants (33–43) metals have distinct advantages particularly when in emulsion/colloidal form. In recent times, silver has been reevaluated in the form of nanoparticles (54–62). The use of small clusters of metals or colloids is not a recent phenomenon, Faraday (1850s) was fabricating colloidal-gold (3-30 nm) and realized that colloidal-gold in the nanometer range has chemical and physical properties not found in bulk gold (63). The reasons for the difference are due to the volume occupied by the electron. In metals, the electrons occupy a large volume, in that the separation between the valence (semi-conductor or insulator-like properties) and conductance (metal-like properties) is negligible, yielding a conducting material, with a very radius. If the separation were to increase or become slightly larger (than kT) semiconductor properties would be observed and further the separation then insulator properties would be observed. These electronic properties depend upon the relationship between the work function and the electron affinity or ionization of the atom or the dielectric constant. As the dimensions are reduced to slightly greater than Bohr radius, the work function is smaller than the ionization energy or electron affinity, the metal clusters are electronically stable because they have a particular separation which affords thermodynamic and electronic stability (e.g. clusters size of 13, 55 and so forth) (64) and large surface area to volume ratio, giving each atom within the cluster and ‘active’ surface for chemical reactions to occur (65), because unlike the bulk metal, the majority of atoms are exposed to the environment, in which the orbital symmetry and energy of each atom facilitate rapid catalysis (66), a fact well known since many catalysts are dispersed as fine powders. Optically reduction in size, will restrict the volume over which the electron moves (below the electron mean free path) resulting in coherent oscillations over the cluster with electromagnetic radiation of light, yielding visible colors (67). These surface plasmon absorption of noble metals have application in (photo)catalysis (68), optoelectronics (69) and storage devices (70), as well as sensors (71) and disinfectants (72). A consequence of this “down-sizing” is that many structural isomers can be constructed using the same number of atoms, many of which have similar binding energies (73), and that the final geometry is strongly dependent on the synthesis procedure (74). Through selective use of solvent polarity, pH, temperature, whether chemical (75) or radiolytic (76) reducing agent are employed, other dispersing agents, speed of mixing, and whether different cluster sizes or range of sizes and shapes (77). Use of zeolites (78) or polymer templates (79) can thus allow one, two and three-dimensional surfaces to be constructed, 135 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

each of which is highly active (80). Their historical use as catalysts in synthesis have been supplemented as disinfectants, nothing that the cluster dimensions can be engineered to be smaller than or equal to the microbe. This has allowed bulk metals such as zinc, copper, silver to be reused as potent nano-bactericidal agents.


Mechanism of Inhibition of Metal/Transition Metal Oxide Based Disinfectants The mode of action of these metals is similar to chemical disinfectants (34–42, 53), with a few subtle differences. Metal ions can diffuse into the membrane and disrupt cell membrane proteins, promoting metal-assisted catalysis of reactive oxygen species, such as hydroxyl radicals, in addition to nucleic acid binding, which is general to metal ions (81). Since the metal species is not consumed, its time of action (or persistence) can be considerably longer than chemical disinfectants such as bleach. In addition, silver is known to irreversibly-bind to cysteine, causing protein denaturation, changes in permeability of the cell and inhibition of respiration through disruption of cytochromes, proton motive force of the membrane and oxidative phosphorylation (82). In the form of silver sulfadiazine, additional wall damage is observed in the form of micro vesicle formation and nucleic acid denaturation. Titania is a transition metal oxide and has been extensively used in water purification and disinfection particularly with Gram-negative microbes through photocatalytic oxidation and generation of reactive oxygen species (ROS), usually coupled with ultraviolet radiation. The role of titania is to provide an active surface for catalysis to proceed and the role of UV to supply sufficient energy to break chemical bonds (83). The role of UV irradiation can be replaced through introduction of a metal center, such as silver, which would supply electrons to the titania to complete the catalysis and generation of ROS (84). Thus fabrication of nanocomposites such as silver-titania would enable disinfectant water under visible light conditions, primarily through ROS-mediated pathways. The formulations of nanodisperant colloids (e.g. silver, titania and other transition metal/metal oxides) had led to a resurgence of interest in their application in water disinfection science (54–62). These ‘second wave’ class of disinfectants has been evaluated against a vast array of microorganisms and appear to be effective at inhibiting microbes. This reevaluation is due to precise control of surfaces (both dimensionally and macroscopically) including formulation of defined optioelectrical and magnetic properties. Lastly, unlike certain chemical disinfectants, these nanocolloid systems have longer persistence and can be effective at much lower doses, thereby minimizing generation of potential disinfection by-products. The mechanism by which inhibition is achieved is multifold but appears to involve cellular disruption either directly (membrane insertion/ change in cellular permeability/ nucleic acid or protein denaturation) or indirectly through generation of reactive oxygen species as summarized in Figure 2. Although a generalization, inhibition may be achieve using four approaches: membrane insertion and lysis of the cell wall (1), metal-catalyzed generation of reactive oxygen species (2) 136 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


which also weaken the cell membrane, leading to protein (star) or nucleic acid (○) denaturation, disruption of membrane respiration enzymes (3) and inhibition of cellular replication through covalent modification of nucleic acid base-pairs (4).These four general methods may be employed towards microbial inactivation based on data from numerous studies (54–62).

Figure 2. Silver atoms or ions may be release (beginning of arrow 4), which may directly interact with phospholipids of the outer membrane, in addition to metal catalyzed generation of radical or reactive species, which cause lipid peroxidation; direct membrane insertion via membrane insertion leading to increased membrane permeability and loss of membrane potential (2); mechanical weakening of the cell wall through complexation of wall proteins, lipids and/or glycans leading to mechanical failure of the wall (1); once migrated into the cytoplasm, additional oxidation of critical proteins (star) and nucleic acids (○) leading to protein denaturation and nucleic acid fragmentation (end of arrow 4); binding to respiratory proteins inhibiting electron transport (3).

The Third Wave: A Historical Overview The basic concept of using structured one-dimensional (colloids) (85), twodimensional (films) (86), three-dimensional (cages, MOFs) (87) without additional supports or with structured supports such as nanotubes (88), chitosan (89), silica (90) and polymers (91) has been established for a wide variety of uses including disinfection and microbial inactivation (92). Structured arrays like metal organic frameworks are central metal ion coordinated within an organic lattice framework. This provides a very high internal surface area, structured internal pores that can retain solvent and enable coordinate between the MOF and microbe surfaces, in addition to the ability of the outer surface separately interacting with the microbe surface. This provides great flexibility and has recently been demonstrated first by 137 In Nanomaterials for Biomedicine; Nagarajan, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


the J. Liu Group in 2010 (87) and separately by another study (93). The former demonstrated using an octatopic framework with a cobalt central atom, while the latter used Ag-based organoboron framework with great promise for disinfection of model microorganism. Metal-MOF structures retains many of the advantages of nanocolloid such as silver-titania, in long term persistence, low doses (compared to chemical disinfectants such as bleach and even other colloids), size control and effectiveness under visible-light conditions. The added advantage of MOFs (elaborated in the following sections) is the ultrahigh surface area (greater than the use of an only a colloid) and not only use of the internal surface area but also the internal MOF area towards disinfection. The three-dimensional lattice can form a ‘web-like’ structure, which can surround the microbe and be effective from all-sides, whereas with colloids, between 20-50 would be required to surround the microbe, here considerably less MOF is required and each MOF exerts greater lethality than a single nanoclusters. For this reason and the structural differences, these are known as the ‘Third wave’ of nanodisinfectants. These motifs built upon are more flexible than zeolites framework of Si/Al (e.g. Na2Al2Si3O10·2H2O) (94). In these type of structures the aluminosilicates occupying tetrahedral geometry (Tn) bridged by oxygen atoms in the form of polygon rings (nMR), which in turn are connected to form larger small (

Historical Overview of the First Two Waves of ... - ACS Publications

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